There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying analytes such as chemical, biochemical, and biological substances. In particular, methods for measuring small quantities of pharmaceuticals, metabolites, biological markers, microorganisms, viruses and other pathogens are desired. The presence of these materials can often be determined by binding methods which exploit the high degree of specificity which characterizes many biological systems. Known methods which rely on binding to detect a molecule of interest present in a sample include nucleic acid hybridization techniques and protein-ligand interactions such as antibody-antigen binding. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence/activation or absence/deactivation of an observable label which has been attached to one or more of the materials comprising the complex.
Sensitivity and selectivity are both desirable attributes of any system for detecting specific molecules of interest present in a sample comprised of a plurality of components. Sensitivity, in DNA hybridization and other bioassays for the detection of biological molecules of interest, is important in clinical diagnostics (Liron and Fisher Eds. Novel Approaches in Biosensors and Rapid Diagnostic Assays, Kluwer Academic/Plenum Publishers: New York, 2000; Kenton et al. 1992, Clin. Chem. 38:873; Chistodoulides et al. 2002, Anal. Chem. 74:3030), forensic chemistry (Heller, 2002, Annu. Rev. Biomed. Eng. 4:129; Nelson et al. 1996, J. Forensic Sci. 41:557), environmental investigations (Lucarelli et al. 2002, Talanta 56:949; Min et al. 2002, Anal. Biochem. 303:186), pharmaceutical studies (Heller, 2002, Annu. Rev. Biomed. Eng. 4:129; Pollice et al. 1985, Clin. Lab. Med. 5:463), and biological warfare agent detection (Smith, 2002, Anal. Chem. 74:462A; Miao and Bard, 2003, Anal. Chem. 75:5825). Thus any system which provides for sensitive and selective detection of molecules of interest will have broad applicability in all of these fields.
Electrochemiluminescence (ECL) methods have been widely used in binding studies, because of their high sensitivity, wide dynamic range, and selectivity (U.S. Pat. No. 6,316,607; Bard, A. J. Ed. Electrogenerated Chemiluminescence, Marcel Dekker New York, 2004). For example, a variety of techniques are available for the detection of DNA, where electrochemical, fluorescent, and ECL active labels attached to a target single stranded DNA (t-ssDNA) produce the measurable signal in the analysis process (Liron and Fisher Eds. Novel Approaches in Biosensors and Rapid Diagnostic Assays, Kluwer Academic/Plenum Publishers: New York, 2000; Yang and Ngo Eds. Biosensors and Their Applications, Kluwer Academic/Plenum Publishers: New York, 2000; Cunningham, Introduction to Bioanalytical Sensors, J. Wiley & Sons, Inc.: New York, 1998). The sensitivity of these methods is often limited since the intensity of the measured signal is generally proportional to the amount of t-ssDNA, and traditionally, only one or a few labels can be attached to one t-ssDNA. A number of approaches have been developed in which one DNA can be labeled with a larger number of labels, so that a higher sensitivity can be achieved (Wang and Merkoci, 2003, Langmuir 19:989; Zhao et al. 2003, J. Am. Chem. Soc. 125:11474). These methods do not provide the sensitivity required to detect quantities in the femtomole (fmol) range. Nor do they provide low non-specific binding, the ability to distinguish between complementary hybridization and a 2 base pair mismatch or multiple measurements.
The need remains, therefore, for highly sensitive detection systems (e.g., in the fmol range) that provide high selectivity and low non-specific binding. The system should have broad applicability so that it can be used to detect virtually any molecule of interest, provided it is capable of binding to or interacting with at least one other molecule (e.g., a specific binding partner). When the molecule of interest is a nucleic acid, e.g., DNA, the system should be able to distinguish between each of the following: complementary hybridization, at least 2-base pair-mismatched hybridization, and non-complementary DNA hybridization.
Ideally, the detection system will provide both a simple treatment to eliminate non-specific binding of the ECL label and high stability of the ECL label thereby allowing for the possibility of taking multiple measurements, without a loss of signal. Each of these needs, at least, is met by certain embodiments of those disclosed herein.